Uracil Excision for Assembly of Complex Pathways

  • Ana Mafalda Cavaleiro
  • Morten T. Nielsen
  • Se Hyeuk Kim
  • Susanna Seppälä
  • Morten H. H. NørholmEmail author
Part of the Springer Protocols Handbooks book series (SPH)


Despite decreasing prices on synthetic DNA constructs, higher-order assembly of PCR-generated DNA continues to be an important exercise in molecular and synthetic biology. Simplicity and robustness are attractive features met by the uracil excision DNA assembly method, which is one of the most inexpensive technologies available. Here, we describe four different protocols for uracil excision-based DNA editing: one for simple manipulations such as site-directed mutagenesis, one for plasmid-based multigene assembly in Escherichia coli, one for one-step assembly and integration of single or multiple genes into the genome, and a standardized assembly pipeline using benchmarked oligonucleotides for pathway assembly and multigene expression optimization.


BioBricks DNA editing Metabolic engineering Molecular cloning Synthetic biology Uracil excision cloning 


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Ana Mafalda Cavaleiro
    • 1
  • Morten T. Nielsen
    • 1
    • 2
    • 3
  • Se Hyeuk Kim
    • 1
  • Susanna Seppälä
    • 1
  • Morten H. H. Nørholm
    • 1
    Email author
  1. 1.Novo Nordisk Foundation Center for Biosustainability, Technical University of DenmarkHørsholmDenmark
  2. 2.Plant Biochemistry Laboratory, Department of Plant and Environmental SciencesUniversity of CopenhagenCopenhagenDenmark
  3. 3.Center for Synthetic Biology, bioSYNergy, University of CopenhagenCopenhagenDenmark

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